枯草芽孢杆菌寡聚—1，6—葡萄糖苷酶基因的克隆及其在大肠杆菌中的表达

本研究用鸟枪法构建了枯草芽孢杆菌（Bacillus subtilis)HB002的基因组文库，经平板法筛选得到了六株能水解合成底物对－硝基苯－α-D－葡萄糖吡喃糖苷的阳性克隆，经鉴定均含克隆了寡聚－1，6－葡萄糖苷酶基因的重组质粒（命名为pHBM001-pHBM006)。选择pHBM003,对其插入片段测序分析，此片段内有一编码561个氨基酸的开放阅读框，该 蛋白质的计算分子量为65.985kD。HB002的寡聚－1，6－葡萄糖苷酶的氨基酸序列与Bacillus sp.和凝结芽孢杆菌（Bacillus coagulans)的寡聚－1，6－葡萄糖苷酶的氨基酸序列一致性分别为81％、67％，相似性分别为89％、79％。从pHBM003中扩增出寡聚－1，6－葡萄糖苷酶基因，克隆到pBV220上，转化大肠杆菌（Escherichia coli)DH5α，得到三个能水解对－硝基苯－α－D－葡萄糖吡喃糖苷的阳性克隆HBM003－1～HBM003－3，将此三个菌株热诱导表达，SDS－PAGE电泳可检测到特异表达的蛋白质，其中HBM003－1、HBM003－2表达的蛋白约66kD，为完整的寡聚－1，6－葡萄糖苷酶，而HBM003－3表达的蛋白质偏小；表达的蛋白质均有寡聚－1，6－葡萄糖苷酶活性。

Cloning and Expression in Escherichia coli of Oligo-1,6-glucosidase Gene from Bacillus subtilis HB002

The gene coding for oligo-1,6-glucosidase of Bacillus subtilis HB002 was cloned by the shotgun-cloning method and sequenced by the chain-termination method of Sanger et al. It consists of an open reading frame of 1683 bp. The amino acid sequence of oligo-1,6-glucosidase deduced from its nuecleotide sequence predicts a protein of 561 amino acid residues with a Mr of 65.985 kD, which is 81% and 67% identical to those of oligo-1,6-glucosidase from Bacillus sp. and Bacillus coagulans, respectively, 89% and 79% similar to those of oligo-1,6-glucosidase from Bacillus sp. and Bacillus coagulans, respectively. The oligo-1,6-glucosidase gene of Bacillus subtilis HB002 was cloned into Escherichia coli expression plasmid pBV220, the result of SDS-PAGE showed that the oligo-1,6-glucosidase gene had been expressed in Escherichia coli DH5 alpha, the expressed oligo-1, 6-glucosidase has enzymatic activity.